Experience with both oxygen and nitric oxide measurement using self-referencing probes, and, in particular, the ability to build our own carbon based microsensors has opened up new horizons for molecular flux detection. The detection of ascorbic acid was explored on the request of Harold Behrman (B00. of Obs. and Gyn. Yale Medical School). Ascorbic acid has the curious quality of exhibiting pronounced extracellular flux patterns, with slow oscillations over extended time periods. This is particularly evident in the mammalian CNS where AA electrochemical signals need to be filtered out for catecholamine detection. Our intention in this sub-project was to explore our ability to produce a self-referencing system able to measure single cell flux and thus contribute to our understanding of the underlying AA transport physiology. These probes are built upon the basic carbon fiber electrode used for the construction of the nitric oxide probes and are modified electrochemically to improve sensitivity for ascorbic acid. This preparation involved the electrochemical treatment of a carbon fiber (1.5V for 3 sec; 70 Hz 3V triangle wave with 1.5V offset for 10 sec; followed by a hold at 0V for 30 minutes). This results in a decrease in the working potential required to oxidize AA from 0.9V to 0.05V. The electrodes are calibrated in solutions of known AA concentrations (linear calibration). While the operation of these microprobes in the self-referencing mode has been verified using an artificial gradient, we have not yet applied them to a biological system. Continued development of the self-referencing electrochemical microsensors can occur through three approaches. These include the development of new sensors, the application of cyclic voltammetry and chronoamperometry, but the most rapid approach is the adaptation of existing electrochemical probes for single cell use. An example of the later approach is the new self-referencing ascorbic acid probe whose development was rapid and simpler than moving from oxygen to NO. We anticipate being in a position to over biomedical researchers a "menu" of probes from which they can choose in designing their experiments.